Improved salvage of complicated microvascular transplants monitored with quantitative fluorometry.

Quantitative fluorometry has been used to monitor circulation in transplanted toes and cutaneous flaps in our unit since 1982. Analysis of 177 uncomplicated transplants monitored by quantitative fluorometry shows that this technique has low false indication rates for arterial occlusion (0.6 percent of patients) and venous occlusion (6.2 percent of patients). None of these patients was reexplored because of a false monitor reading, and except for single abnormal sequences, monitoring appropriately indicated intact circulation throughout the postoperative period. Quantitative fluorometry has correctly indicated vascular complications in 21 (91.3 percent) of 23 transplants over an 8-year period. The salvage rate (85.7 percent) of the fluorescein-monitored reexplored transplants was significantly higher than the salvage rates of similar reexplored transplants not monitored with fluorescein and of reexplored muscle flaps (which cannot be monitored with the fluorometer used at this unit). These clinical data indicate that quantitative fluorometry is a valid and useful postoperative monitor for transplanted toes and cutaneous flaps.     

Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli.

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.     

Reduction of nitroaromatic compounds by anaerobic bacteria isolated from the human gastrointestinal tract.

Human intestinal microbial flora were screened for their abilities to reduce nitroaromatic compounds by growing them on brain heart infusion agar plates containing 1-nitropyrene. Bacteria metabolizing 1-nitropyrene, detected by the appearance of clear zones around the colonies, were identified as Clostridium leptum, Clostridium paraputrificum, Clostridium clostridiiforme, another Clostridium sp., and a Eubacterium sp. These bacteria produced aromatic amines from nitroaromatic compounds, as shown by thin-layer chromatography, high-pressure liquid chromatography, and biochemical tests. Incubation of three of these bacteria with 1-nitropyrene, 1,3-dinitropyrene, and 1,6-dinitropyrene inactivated the direct-acting mutagenicity associated with these compounds. Menadione and o-iodosobenzoic acid inhibited nitroreductase activity in all of the isolates, indicating the involvement of sulfhydryl groups in the active site of the enzyme. The optimum pH for nitroreductase activity was 8.0. Only the Clostridium sp. required added flavin adenine dinucleotide for nitroreductase activity. The nitroreductases were constitutive and extracellular. An activity stain for the detection of nitroreductase on anaerobic native polyacrylamide gels was developed. This activity stain revealed only one isozyme in each bacterium but showed that the nitroreductases from different bacteria had distinct electrophoretic mobilities.     

Whether cytochrome p450 induction accompanies glucuronosyl and glutathione transferase induction by isomers of dipyridyl appears unrelated to dose and iron chelation properties

Administration of 4, 4′dipyridyl to rats induces the activities of xenobiotic transferases (phase II drug metabolizing enzymes), UDP-glucuronosyl-tranferase and glutathione-S-transferase, and also the concentration and activity of cytochrome P450 (a phase I drug metabolizing enzyme). 2, 2′Dipyridyl, an isomer possessing iron chelation properties, only induces the phase II enzymes. Although the magnitude of the phase II induction by 2, 2′dipyridyl increases with increasing dosages, the selective induction of only phase II activities remains inviolate. Co-administration of 2, 2′dipyridyl does not prevent 4, 4′dipyridyl from inducing cytochrome P450, suggesting that the iron chelation property is not the factor that precludes 2, 2′dipyridyl from coordinately inducing cytochrome P450 with the transferases.     

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.     

Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon.

In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.     

Chi-Stimulated Recombination between Phage λ and the Plasmid λdv

Chi promotes Rec-mediated recombination between phage lambda DNA and the homologous plasmid lambda dv. In the absence of Chi, some of the interactions splice lambda dv into lambda, whereas others patch information from lambda dv into lambda. When Chi is in the phage DNA, splices and patches are increased in frequency by the same factor. This result strengthens the analogy between Chi and recombination-promoting elements in fungi. It also rules out one model for the previously reported orientation dependence of Chi phenotype.     

Ethidium bromide: a nucleic acid stain for tissue section

The phenanthridinium dye, ethidium bromide (EB), selectively intercalates into double-stranded regions of nucleic acids with a large and specific increase in fluorescence. When used for the staining of fixed tissue sections, the dye stains cellular nuclei with excellent resolution of microscopic detail. In some fixed tissues, particularly pancreatic acini, cytoplasm stains intensely and this staining can be abolished by digestion with trypsin and ribonuclease. The orange fluorescence of EB can be easily distinguished from the green fluorescence of fluorescein and EB is thus an excellent counterstain for immunofluorescence. Ethidium bromide is a useful and practical stain for the fluorescence microscopy of tissue sections and, in combination with enzymatic digestion of RNA, provides a simple way to differentially localize DNA and RNA.     

Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages

Human immune interferon preparations have anticellular activity on human cell lines (WISH and HEp-2). This anticellular activity copurified with the human immune interferon and appears to be a function of the immune interferon molecule. On the basis of a unit of antiviral activity, purified human immune interferon had about 20 and 100 times more anticellular activity than purified fibroblast or leukocyte interferon, respectively. The possible implications of this finding in the treatment of human neoplasia are discussed.